Team:Valencia UPV/Notebook

Valencia UPV iGEM 2015

Notebook

CONSTRUCTIONS


5 June 2015

We had 2 cultures from the last day, corresponding to other 2 colonies of ligation.

Agrobacterium culture of promoter less: Luciferase + Renilla

Minipreps

Digestion with BamHI and EcoRV

Agarose gel 1%

How to ask and make primers?

  • Select the sequence to amplify and save in FASTA format.
  • gbCloning, go to Tools-Domesticator-1º Category
  • Add FASTA and select parts.
  • On the protocol we have the primers
  • The oligos they give us:
    • 4 first nucleotides: so the enzyme can recognize without problems
    • 6 following bingind sites.
    • 1 extra nucleotide.
    • 4 overhangs.

Meeting with Daniel Ramón (Biopolis).

Ligation with part 2 and 24 of task sheet.

PIF6 + PhyB; ?1Etr8 CMV_Bxb1_T35S
1µL 892 (PIF α1)1µL 1097 (Etr8 CMV) Pupd2
1µL 88E (Phy α2)1µL Bxb1 (PuPD)
1µL ?1 1µL Tnos PuPD
1.2µL Buffer ligase1µL α1
1µL Bsmb15.8µL H2O
6.8µL H2O

If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb.

If we make a digestion of 896 (Luc:PIF6:PhyB)with EcoRV, we obtain: 11608, 3942 pb.


June 2015

Transform to E.coli from PIF+Phy and Bxb1

  • 1.5µL of ligation
    • Cuvette on ice
    • Competent cells + 1.5µL of ligation
    • Pulse (electroporator) at 1500V
    • Add 300µL shock medium and put Eppendorf 1h at 37ºC

Culture on petri dishes the ligations.

Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI.

Agarose gel.


7 June 2015

We’ve got white colonies! (from PIF+Phy and Bxb1)

Pick two colonies from each construction.

4 tubes

  • 3.5µL LB each tube

2) 2 tubes + 3.5µL Kanamycin (K)


8 June 2015

Minipreps of the 4 liquid cultures and digestion to see the band patterns.

Digestion:

Etr8(CMV):Bxb1:Tnos; ?1EcoRI6345, 238
EPIF6 + PhyB-PV16; ?1BamHI6686, 1439, 2685, 2237

Agarose gel was made:

Bxb1 (C1)Bxb1 (C2)EPIF6 + PhyB-PV16 (C1)EPIF6 + PhyB-PV16 (C2)
okok

Repeat digestion (errors).

We don’t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the colony 2 has better bands pattern.

Optimized ligation of PIF-Phy-Lac-Renilla-P19

  • 1 µL vector
  • 0.8 µL dilution ½ 160
  • 1.7 µL big part
  • 1.2 µL BSA
  • 1.2 µL buffer
  • 1 µL BsbmI
  • 1 µL ligase
  • 4.15 µL H2O
  • Ratio 1:2 vector insert

As Bxb1 was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at 37ºC to glycerinate later.

We design primers to binding domain (BD) and PIF.

  • Problem: domesticator is introduced in an old pUPD. The new one has different bases.
  • Change manually the pUPD bases in the program (Benchling).

9 June 2015

Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)

EPIF6-PhyB-VP16PvuII (green buffer)3663, 9472pb

Agarose gel 1%:

EPIF6-PhyB-VP16 (C1)EPIF6-PhyB-VP16 (C2)EPIF6-PhyB-VP16 (C3)
nonono

We see three bands: 7000, 4000, 1900pb

Transform optimized ligation (yesterday 8/6)

  • 3.5 µL ligation product (EPIF6-PhyB-VP16 + Luciferase + Renille + P19)
  • 40 µL electrocompetent cells.
  • Pulse of 1500V
  • Store 1h at 37ºC
  • Plate culture at agar with Spectinomycin: 50 µL of the transformation.

10 June 2015

  • Check the primers and order LexA, Gal4, PIF6, LacI, Dronpa.
  • Check linker VP16 (88E) and make a primer for it.
  • Take out glycerinate of Ω2.

Alfredo’s part is not working.

  • Pick colonies of transformation and make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).
  • Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6
  • Digestion:

PIF + Phy:VP16 PvuII (buffer green 10x) 3663, 9472

PIF + Phy:VP16 BamHI 1939, 2685, 2337, 6674

  • Agarose gel 1% (10 samples, 8 + 2 molecular markers)

PIF + Phy (PvuII) C3 PIF + Phy (PvuII) C4 PIF + Phy (PvuII) C5 PIF + Phy (PvuII) C6

no ok no No

PIF + Phy (BamHI) C3 PIF + Phy (BamHI) C4 PIF + Phy (BamHI) C5 PIF + Phy (BamHI) C6

no ok no No

  • Transformation of Agrobacterium (C58) of the 896 construction (EPIF6-PhyB-VP16 + luciferase). We are not going to have the positive control and we won’t be able to quantify (we don’t have Renilla + P19).

11 June 2015

  • Minipreps of the culture:
  • Digestion:

E:PIF6:PhyB:VP16:luc:ren BamHI 4209, 3756, 6100, 6674

EcoRV 3942, 2989, 2475, 381, 10952

Gel:

PIF6:PhyB:VP16:luc:
ren C1 (BamHI)PIF6:PhyB:VP16:luc:
ren C3 (BamHI)PIF6:PhyB:VP16:luc:ren
C1 (EcoRV)PIF6:PhyB:VP16:luc:ren
C3 (EcoRV)
??

Transformation in Agrobacterium of Renilla (160) due to that we could not join this with PIF:phyB and so we will do a cotransfection of both plasmids. Make petri dish culture with kanamicyn and rifampicin.


12 June 2015

The petri dish with PIF:phy:luc was taken out the 37ºC room and put into the fridge to pick colonies tomorrow.


13 June 2015

  • Pick colonies to make liquid culture:
    • Renilla in agrobacterium: just one colony, it was made liquid culture but check carefully the gel.
    • It was noticed that the piece 160, renilla, needs a pSub plasmid to replicate itself so we will transform 160 into a agrobacterium with this plasmid (C58 pSub).
  • Ligation:

BxbI; alfa1+phyB; alfa2

1µl BxbI

1 µl phyB

1 µl ?2

4.6 µl H2O

  • Transform renilla (160) into agrobacterium and make

15 June 2015

  • Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was ?2

BxbI + 35S:E-PIF6:tnos; ?1

1µl BxbI

1 µl phyB

1 µl ?1

4.6 µl H2O

  • KDronpa has arrived:
    • Centrifuge it 2-5sec at max velocity.
    • Add 50 µl to have a concentration of 20ng/µl
    • Mix it with the vortex and spin.
  • Ligation: en la libreta pone que se liga a pUPD2

KDronpa; pUPD2

1 µl KDronpa

1 µl pUPD2

5.6 µl H2O

  • It was not possible to pick colonies of the Agrobacterium transformed because they did not grow. Maybe the problem is that with tetraciclyn bacterias grow slowly. Wait 1 day more.
  • Transformation of the ligation, BxbI + 35S:E-PIF6:tnos; ?1, into E.coli.
  • Make an agar culture in petri dish and let grow 16h at 37ºC.

16 June 2015

  • Transformation of the ligation, KDronpa, into E.coli.
  • Pick colonies of BxbI:E-PIF6 and make liquid culture (C1-C3).
  • Primers had arrived, it has been done the resuspension (dilution 1:10)of all of them.
PrimersCode TemplateWorking temperature? ºC
LacI F1LacI (858)69.7
LacI R 2
Gal4 F3We did not take out the glicerynate.63.2
Gal4 4
LexA F5LexA (732)62.7
LexA R6
PIF:VP16 F7PIF6 (288)60.1
PIFVP16 R8
NDronpa F19Kdronpa67.7
NDronpa R110
Dronpa F21158.5
NDronpa R212
  • A PCR with all the primers and the fragments was done, the samples were put in order with the temperature.
    • The templates were in dilution 1:50, exception of KDronpa that was dilution 1:5 and the primers 1:10.
PCR Fusion Taq (50µl)
DNA template (10 µg/µl)
0.5 µl fusion taq
2.5 µl primer F
2.5 µl primer R
2 µl NTPs
31.5 µl H2O
17 June 2015
  • Pick colonies and make liquid culture of:
    • KDronpa (C1-C4)
    • Ligations with the PCR’s products:
    • Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16, 9+10, 11+12.
Template PCR; pUPD2
0.5µl template
1µl pUPD2
6.1µl H2O
  • Minipreps of liquid cultures:
    • BxbI:E-PIF6 (C1-C3)
  • Agarose gel with the PCRs:

Template 1+2 5+6 7+8PIF 7+8VP16 9+10 11+12

Band pattern 1017 284 391 478 464 290

Gel result ok ok ok ok No DNA ok

  • Transformation in E.coli of the correct ligations:
    • 1+2, 5+6, 7+8PIF, 7+8VP16, 11+12
    • Put in cloranfenicol petri dishes.

18 June 2015

  • Minipreps of the liquid cultrures:
    • KDronpa (C1-C4)
  • Digestions:

KDronpa EcoRI 2800

  • Gel:

Kdronpa C1 Kdronpa C2 Kdronpa C3 KdronpaC4

no no ok no

Etr8:BxbI:phyB C1 Etr8:BxbI:phyB C2 Etr8:BxbI:phyB C3

No no no

We discovered that the construction with BxbI did not go well because our lab college gives us the wrong piece.

  • Take glicerynates out:
    • Gal4; pUPD (731)
    • ?2
    • PromotersinATG (GB00552)
    • Renilla (160)(159)(109)
  • PCR:

NDronpa

2.5 µl (9+10) primer F

2.5 µl (11+12) primer R

2 µl NTPs

0.2 µl Taq

10 µl Buffer

31.5 µl H2O

  • Ligations:

Etr8:BxbI:T35S; α1 Template PCR; pUPD2

1 µlEtr8 0.5µl template

1 µl BxbI 1µl pUPD2

1 µl T35S 6.1µl H2O

5.8 µl H2O

Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16


19 June 2015

  • We do a PCR with the normal Taq polymerase.
    • 1µl of DNA’s template (9+10, 9+12 and 11+12)
    • 2µl of specific buffer
    • 2µl of NTPs
    • 1µl primer forward
    • 1µl primer reverse
    • 0.5 µl of Taq
    • 12.5 µl H2O
    • These quantities multiplied by 3.
  • Minipreps of the yesterday’s glycerinated cultures.
  • To do the digestions we add in each eppendorf.

Minipreps: Enzime Band pattern

159 pDGB1_?2 renilla EcoRV 2909, 2475,882, 812, 381

Entry vector, ?2 EcoRV 6652, 621

552 pP35s NoATG, pUPD EcoRI 2997, 1090

160 renilla pDGB1, a2 EcoRV 4601, 2475, 381

731 pUPD pGal4BD (CDS) EcoRI 2997, 2493

109 GB1_a1 355:renilla:Tnos EcoRI 2580, 2493

  • We make an agarose gel with the digestions made before and the PCR of KDronpa.

159 160 ?2 552 731 109 9+10 9+12 11+12

ok ok ok ok ok ok no ok ok

  • Transformation of the ligations:
    • 50 µl of electrocompetent cell
    • 1.5 µl of ligation
    • Put 1 min before the cubettes in ice
    • Make the transformation (1500V)
    • Put the transformed cells 1h at 37ºC
  • We made an stack of Cloranfenicol petri dishes
    • 250ml LB aga
    • X-gal (1:500): 500 µl
    • Iptg (1:1000): 250 µl
    • Cloranfenicol (1:2000): 125 µl

20 june 2015

We have White colonies of renilla! Also of Etr8 + Bxb1

We have also pUPD colonies but they are so close to the blue ones that we can’t pick anyone.So we make strakes in another plates to have the colonies separated.

  • We make a liquid culture of Agro of Renilla.
    • 5ml of LB agar
    • 5 µl rifampicine
    • 5 µl of kanamicine
    • 5 µl tetracycline
    • Incubate for 2 days (48h) at 28ºC.

21 June 2015

  • Pick colonies of and put into a liquid medium of 3 ml of LB agar, 3 µl of each antibiotic (kanamicine, spectomicine, cloranfenicol):
    • Plates (17/06/15): PIF (C1, C2) (with cloranfenicol), VP16 (C1, C3) (with cloranfenicol), LacI (C1, C2, C3) (with cloranfenicol)
    • Plates (19/06/15): Bxb1 (C1, C2, C3) (with kanamicine), VP16 (C4, C5) (with cloranfenicol), LacI (C1, C2) (with C1, C2) (with cloranfenicol), PIF (C1, C2, C3, C4, C5) (with cloranfenicol), LexA (C1, C2) (with cloranfenicol).
  • We take out two glicerynates of GFP and BFP (of the Alfredo’s box)

22 June 2015

  • We made minipreps of the liquid culture of the day before:
    • LacIBD, pUPD (C1-C5)
    • LexABD, pUPD (C1, C2)
    • Etr8(CMV):Bxb1 (C1-C3)
    • PIF6,pUPD (C1-C5)
    • VP16, pUPD (C1, C4, C5)
  • Make the digestions of all the minipreps:

LacIBD, pUPD NotI 2046, 1053

LexABD, pUPD NotI 2046, 321

Etr8(CMV):Bxb1 NotI 1532, 1290, 5896

PIF6,pUPD NotI 2046, 407

VP16, pUPD NotI 2046, 500

  • Viral system:……….
  • We received the reported Bxb1!
    • 500ng of sample
    • Centrifuge at 3000rpm for 5 seconds (spin).
    • Add 50 µl H2O
    • Shake it and let at 50ºC for 20min
    • Make a PCR of Gal4 and NDrompa (9-10), the primers of NDrompa are aliquoted
  • …..

Make the gel with all the digestions writed before.

Lac1Lac2Lac3Lac4Lac5Lex1Lex2Bxb1,1Bxb2, 2Bxb1,3
Okokokokoknonookokno
PIF1PIF2PIF3PIF4PIF5VP16,1VP16,4VP16,5
Nono-okokokokok
PCRS………
  • We make ligations of:
    • Etr8(CMV):BxbI in a1 + PhyB:P16 in a2, ?1
    • LacIBD in pUPD2 + PIFBDless+promoter+terminator, a1
    • KDrompa in pUPD2 + LacBD+promoter+terminator, a1
    • Gal4BD, pUPD2
    • Reporter of BxbI, pUPD2
  • Tomorrow we have to take out pUPD of constitutive promoters, terminators and GFP (CDS).

23 June 2015

  • Transformations in E.Coli of the 5 ligations done yesterday and two more transformations of 5+6(1) and 5+6(2) which are the ligations in pUPD of the 18/06.
  • We put 50 µl of the transformations in the plates. Put in 37ºC.
    • Etr8(CMV):Bxb1 in a1 + PhyB:P16 in a2, ?1
    • LacIBD in pUPD2 + PIFBDless+promoter+terminator, a1
    • KDrompa in pUPD2 + LacBD+promoter+terminator, a1
    • Gal4BD, pUPD2
    • Reporter of Bxb2, pUPD
    • LexABD (5+6(1)), pUPD
  • We have taken out of the -80ºC fridge the glycerinate of GFP, pUPD/ampicilina/GB0059.
  • The liquid culture of Renilla (rifampicina/kanamicia/tetraciclina) doesn’t grow before the two days required. So we decide to put two new colonies in une tube with the three antibiotics and another with rifampicina and kanamicine. Asun say that the tetracycline slow down the growth of Agro.
  • The 4 liquid cultures of LexA+IPTG/+gal are all blue: throw them.
  • We ordered again the primer nº10 (NDronpa R1). Changing one codon in 3’ and delete another in 5’.

24 June 2015

Pick colonies of the plates done yesterday and pass them into a liquid media: 3 µl of LB, 3 µl of antibiotics (K, Spect, Clor).

  • LacIBD+PIF, a1 (C1, C2)
  • Gal4BD, pUPD2 (C1)
  • RepBxb1, pUPD2 (C1, C2, C3)
  • LacIBD+KDonpa, a1 (C1, C2)
  • Etr8(CMV)+Bxb1+phyB+VP16, ?1 (C1)
  • LexABD1, pUPD (C1-C4)
  • LexABD2, pUPD. No colonies.

The viral systems cultures of Agro for the color mosaics are ready before 2 days at 28ºC. We can make the Agroinfiltration.

Buffers of Agroinfiltration:

  • First we have to prepare and ajust the pH of the buffer MES and the buffer MgCl.
  • MES (10x), 100nM; ph=5,6 (adding NaOH). Make 1L.
  • MgCl (100x), 1M. Make 100ml.
  • Solution to agroinfiltration: 10ml of MES(10x) + 1ml of MgCl (100x) + 100 µl of DMSO+acetosiningona and finally “enrasar” to 100ml.
  • 19.6mg of acetosiningona for 500 µl of DMSO
  • Ligation:

ETR8(CMV):Bxb1(a1)+phyB+VP16(a2); ?1 Gal4BD(pcr) + pUPD2

1.5 µl etr8:Bxb1 1 µl Gal4 PCR

1.5 µl 88E (phyB+VP16) 1 µl pUPD2

1 µl ?1 1.2 buffer T4 ligase

1.2 µl buffer T4 ligase 1 µl ligase

1 µl ligase 1.2 µl BSA

1.2 µl BSA 1 µl BsmbI

1 µl BsmbI 5,6 µl H2O

3.6 µl H2O

Quantification of DNA:

  • pUPD GFF (0059): 249 ng/µl
  • ?2: 238 ng/µl
  • Alfredo’s pUPD2, domesticator: 102 ng/µl
  • iGEM704: 405 ng/µl
  • IGEM735: 403 ng/µl
  • 552 AMP 35S noATG: 45 ng/µl
  • PIF (C5), pUPD2: 119 ng/µl
  • pD6B3, ?2 (22/06): 158 ng/µl
  • LacIBD (C1), pUPD (22/06): 129 ng/µl
  • 109k renillaDC: 49 ng/µl
  • IGEM 534: 13.6 ng/µl
  • VP16 (C1), pUPD2:102 ng/µl
  • IGEM 1097: 409 ng/µl
  • K-donpa (C3), pUPD2 (18/06): 174 ng/µl
  • IGEM 858: 487 ng/µl
  • 731AMP Gal4 (19/06): 81 ng/µl
  • IGEM pUPD2 domesticator: 87 ng/µl
  • PIF+phy8 (c1) (08/06): 108 ng/µl
  • 160 renilla, a2 (19/06): 46 ng/µl
  • 159 renilla, ?2 (19/06): 149 ng/µl
  • Etr8:Bxb1 (C1)(22/06): 149 ng/µl
  • IGEM 732: 422 ng/µl

Measurement of the OD:

  • first we have to

25 June 2015

Minipreps of the liquid culture:

  • We don’t observed growth in LacIBD+PIF and LaciBD+K-donpa.

Digestion of the minipreps and do the gel:

Gal4BD, pUPD2NotI2046, 282
RepBxb1, pUPD2NotI2046, 460
Etr8(CMV):Bxb1 + phyB,a1BamHI6674, 2237, 2806, 1174
LexABD, pUPD2NotI2046, 321
9+10, pUPD2NotI464

Gel:

Etr8:Bxb1Lex1Lex2Lex3Lex4Rep1Rep2Rep3Gal4 C1PCR 9+10
nononononookokoknook
  • We make a PCR of the Fusion Taq pH (proof-reading) to prove that the primer received number 10. This new one works! Amplify the sequence of N-donpa (R1).
  • Refresh the cultures of Agro (28ºC). We pick 7.5 µl of the previous culture. And put into a new liquid media with Rifampicine and Kanamicine for 2 days and 28ºC.
  • Ligations:

N-dronpa Rep GFP Gal4 LexA

1 µl PCR 9+10 1 µl Rep Bxb1 1 µl PCR 3+4 1 µl PCR 5+6

1 µl PCR11+12 1 µl promoter without ATG 1 µl pUPD2 1 µl pUPD2

1 µl pUPD2 1 µl Tnos

1.2 µl buffer ligase 1.2 µl buffer ligase 1.2 µl buffer ligase 1.2 µl buffer ligase

1.2 µl BSA 1.2 µl BSA 1.2 µl BSA 1.2 µl BSA

1 µl BsmbI 1 µl BsaI 1 µl BsmbI 1 µl BsmbI

1 µl T4 ligase 1 µl T4 ligase 1 µl T4 ligase 1 µl T4 ligase

4,6 µl H2O 3,6 µl H2O 5,6 µl H2O 5,6 µl H2O

1 µl a2

Etr8:Bxb1+phyB

1 µl Etr8:Bxb1

1 µl 88E

1µl ?1

1.2 µl buffer ligase

1.2 µl BSA

1 µl BsmbI

1 µl T4 ligase

3,6 µl H2O

We transform the ligations in E.Coli ant pass them into agar plates with cloranfenicol for all of them except the ligation of Etr8:Bxb1+phy that goes with bhnfnfg


26 June 2015

We do again the two ligations that didn’t gone?

Rep GFPLacI BD+PIF6
1 µl Rep Bxb11 µl LACIBD, pUPD
1 µl promoter without ATG1 µl PIF6, pUPD
1 µl Tnos1 µl promoter
1.2 µl buffer ligase1 µl T35
1.2 µl BSA1 µl a1
1 µl BsaI1.2 µl buffer BsaI
1 µl T4 ligase1.2 µlBSA
2.6 µl H2O1 µl BsaI
1 µl a21 µl ligase
1µl GFP (0059)2.6 µl H2O

We make a gel of LAcI+PIF6 (C1 and C2). Both of them present the fragment of the vector at 6000 pb but none of them at 2000bp which is the insert one.

LacIBD+PIF; a1EcoRI6345, 1997, 641
LacIBD+PIF C1LacIBD+PIF C2
nono

Measurement of the ODs of phyB+PIF+luc and renilla+P19.

  • phyB+PIF+luc: o.35 (1:2)
  • Ren+P19: 0.34 (1:2)
  • Final volume= 2
  • CCo= 0.35*2
  • CCf= 0.2
  • Ecuation=> Vo*CCo=Vf*CCf
  • So we obtain that Vo(LacI+PIF6)=1.429 µl and Vo(ren)= 1.412 µl.
  • Ligation of:

LacIBD,pUPD + K-donpa, pUPD

1 µl 35S

1 µl LacIBD,pUPD2

1 µl K-dronpa, pUPD

1 µl T35S

1 µl a1

1.2 µl buffer T4 ligase

1 µl BsaI

1 µl T4 ligase

2.6 µl H2O

  • We make the agorinfiltration of (…..) to start checking if the plant react to the different ..

27 June 2015

Transformation in E.coli of LacIBD+K-Dronpa, a1

We Put into plates LexABD and Etr8(CMV):Bxb1:GFP again and also LAcIBD+K-Dronpa.

We make liquid culture of:

  • RepBxb1:GFP (C1-C4)
  • LacIBD+PIF (C1-C5)
  • N-Dronpa (C1-C4)
  • Gal4BD (C1-C5)
  • LexA

28 June 2015

We do the minipreps of the liquid cultures that have grown.

  • RepBxb1:GFP (C1 and C2)
  • LacIBD+PIF (C1-C4)
  • N-Dronpa (C1-C4)
  • Gal4BD (C1-C5)
  • LexA: didn’t grow

Do the digestions of the minipreps:

LacIBD+PIF, a1EcoRI6345, 1997, 641
RepBxb1:GFP, ?2HindIII6345, 2683
Gal4BD; pUPD2NotI2681, 644
N-Dronpa; pUPD2NotI2046, 744

Make the gel.

RepBxb1:GFP C1RepBxb1:GFP C2LacIBD+PIF C1LacIBD+PIF C2LacIBD+PIF C3LacIBD+PIF C4
nononononoNo
Gal4BD C1Gal4BD C2Gal4BD C3Gal4BD C4Gal4BD C5N-Dronpa C1
okokokokokok
N-Dronpa C2N-Dronpa C3N-Dronpa C4
nookok

Take glycerinated:

  • GB0030: p35S
  • GB0036: T35S
  • Make liquid culture of LexABD (C1-C4).
  • We transform again LacIBD+K-Dronpa and RepBxb1:GFP, adding to the agar plates 100 µl of each transformation.

29 June 2015

Do the minipreps of the 4 colonies of LexABD and both glycerinates, 35S and T35S.

Do the digestion of the 4 colonies of LexA and both glycerinates:

LexABD; pPPD2NotI2358, 312
35S; pUPD2NotI2981, 1074
T35S; pPUD2NotI2981, 304

Make the gel:

LexA C1LexA C2LexA C3LexA C4P35ST35S
okokokokOk?Ok?

Make ligations:

LacIBD+K-Dronpa+promoter+termi; a1Gal4BD+K-Donpa+prom+ter; a1LexABD+K-Dronpa+prom+term; a1
1 µl LacI, pUPD21 µl Gal4, pUPD21 µl Gal4, pUPD2
1 µl K-Dronpa, pUPD21 µl K-Dronpa, pUPD21 µl K-Dronpa, pUPD2
1 µl 35S (GB0030)1 µl 35S (GB0030)1 µl 35S (GB0030)
1 µl T35S (GB0036)1 µl T35S (GB0036)1 µl T35S (GB0036)
1.2 µl buffer ligase1.2 µl buffer ligase1.2 µl buffer ligase
1.2 µl BSA 10x1.2 µl BSA 10x1.2 µl BSA 10x
1 µl BsaI1 µl BsaI1 µl BsaI
1 µl ligase T41 µl ligase T41 µl ligase T4
2.6 µl H2O2.6 µl H2O2.6 µl H2O
1 µl a11 µl a11 µl a1
N-Dronpa+VP16; a2Gal4BD+PIF6; a1LacIBD+PIF6; a1
1 µl N-Dronpa, pUPD21 µl Gal4BD, pUPD21 µl LacIBD, pUPD2
1 µl VP16, pUPD21 µl PIF6, pUPD21 µl PIF6, pUPD2
1 µl 35S (GB0030)1 µl 35S (GB0030)1 µl 35S (GB0030)
1 µl T35S (GB0036)1 µl T35S (GB0036)1 µl T35S (GB0036)
1.2 µl buffer ligase1.2 µl buffer ligase1.2 µl buffer ligase
1.2 µl BSA 10x1.2 µl BSA 10x1.2 µl BSA 10x
1 µl BsaI1 µl BsaI1 µl BsaI
1 µl ligase T41 µl ligase T41 µl ligase T4
2.6 µl H2O2.6 µl H2O2.6 µl H2O
1 µl a21 µl a11 µl a1
LexABD+PIF6; a1
1 µl LexABD, pUPD2
1 µl PIF, pUPD2
1 µl 35S (GB0030)
1 µl T35S (GB0036)
1.2 µl buffer ligase
1.2 µl BSA 10x
1 µl BsaI
1 µl ligase T4
2.6 µl H2O
1 µl a2
  • Transform all the ligations into E.Coli.Gal4BD+K-Dronpa and LacIBD+K-Dronpa goes wrong and we have to do it again.
  • Put into a plate the colonies before let stay them 1h at 37ºC.

Quantification of DNA:

  • RepBxb1:GFP (C1): 163.8 ng/µl
  • N-Dronpa, pUPD2 (C4):113.1 ng/µl
  • N-Dronpa (C3): 83.2 ng/µl
  • NDonpa (C1): 116.6 ng/µl
  • Gal4BD (C1): 95.2 ng/µl
  • Gal4BD (C2): 120.7 ng/µl
  • RepBxb1:GFP (C2): 170.6 ng/µl
  • RepBxb1 (C1): 80.6 ng/µl

30 June 2015

Transform Gal4+K-Dronpa and LacI+K-Dronpa

Miniprep of:

  • RepBxb1+GFP (C1-C3)
RepBxb1+GFPHindIII6345, 2683

Gel:

RepBxb1+GFP C1RepBxb1+GFP C2RepBxb1+GFP C3
Nonono

We pick more colonies and make other liquid cultures.

Save the last samples of the leaves of the plants that were under the first experiment, next to the glycerinates in the freezer (-80ºC).

Put in plates the transformations of Gal4+K-Dronpa and LacI+K-Dronpa (50 µl of bacteria in SOC medium).

Make the gel:

  • Add to the digestions 2 µl of loading buffer (6x) because for 5 µl of digestion we put 1 µl of the buffer.

Make liquid culture of two colonies for each plates of the transformations done yesterday, 10 tubes in total.

Take out a glycerinate 35S:Luciferase:Tnos (GB0227) and do a miniprep.


1 July 2015

Do the minipreps of the 10 liquid cultures.

Do the digestions:

LexABD+K-Dronpa;a1EcoRI6345, 2296
N-Donpa+VP16; a2HindIII6345, 2427
Gal4BD+PIF6; a1EcoRI6345, 1867
LacI+PIF; a1EcoRI6345, 2638
LexABD+PIF6; a1EcoRI6345, 1906

Do the gel:

Gal4+PIF C1Gal4+PIF C2LexA+PIF C1LexA+PIF C2LexA+KDronpa C1
okokokokOk
LexA+Kdronpa C2LacI+PIF C1LacI+PIF C2Ndonpa+VP16 C1Ndronpa+VP16 C2
okokokokok
  • Prepare liquid culture of:
  • LexA+PIF; ?1 (C1)
  • LacI+PIF; ?1 (C1)
  • LexA+K-Dronpa (C1)
  • Gal4+PIF; ?1 (C1)
  • VP16, pUPD2 (C1)
  • LexABD, pUPD2 (C2)
  • PIF6, pUPD2 (C5)
  • LacIBD, pUPD2 (C1)
  • Pick colonies and make liquid culture of:
    • Gal4+K-Dronpa (C1 and C2)
    • LacI+K-Dronpa (C1 and C2)
    • RepBxb1+GFP (C4-C6)
  • We sent to sequence:
    • Code:
    • 210.08-249: pUPD2, KDronpa C3
    • 210.08-250: pUPD2, NDronpa C1
    • 210.08-251: pUPD2, NDronpa C3
    • 210.08-252: pUPD2, NDronpa C4
    • The solution have: 10µl of miniprep + 5µl (dilution 1:3) of primers.
  • Data of the luciferase essay with the sample plat RED at 24h (replica2): we obtain a value of 7.4E6.

2 July 2015

Minipreps of:

  • Gal4+K-Dronpa (C1 and C2)
  • LacI+K-Dronpa (C1 and C2)
  • RepBxb1+GFP (C4-C6)
Gal4+K-DronpaEcoRI6345, 3028
LacI+K-DronpaEcoRI6345, 2257
RepBxb1+GFPHindIII6300, 2400

Do the gel:

LacI+K-Dronpa C1LacI+K-Dronpa C2Gal4+K-Dronpa C1Gal4+K-Dronpa C2
??
RepBxb1+GFP C4RepBxb1+GFP C5RepBxb1+GFP C6

Luciferase essay:

  • During the whole experiment we lost three samples: T16/FarRed/1; T24/Red/2 and T0/FarRed/1

Results:

Things to keep in mind for the next experiment:

  • The luminimeter (machine to measure the luminescence) has to be ready before start adding the reactants to the samples because it needs 10min to be ready.
  • Set the timer (10min) with the first sample of luciferase and add the reactant to the other samples as quick as possible.
  • We have to let the renilla stay before putting it in the luminimeter the same time as the luciferase, in this case 15min because with the luciferase we didn’t manage well the time. Theoretically we have to wait 10min.

Make ligations:

LexA:Kdonpa+N-Dronpa; ?1LacI:Kdronpa+N-Dronpa; ?1Gal4:Kdronpa+N-Dronpa; ?1
1 µl LexA:Kdronpa1 µl LacI:Kdronpa1 µl Gal4:Kdronpa
1 µl N-Dronpa1 µl N-Dronpa1 µl N-Dronpa
1 µl ?11 µl ?11 µl ?1
1.2 µl buffer ligase1.2 µl buffer ligase1.2 µl buffer ligase
1.2 µl BSA1.2 µl BSA1.2 µl BSA
1 µl BsmbI1 µl BsmbI1 µl BsmbI
1 µl BsaI1 µl BsaI1 µl BsaI
4.6 µl H2O4.6 µl H2O4.6 µl H2O
LexA:PIF+PhyB:VP16; ?1LacI:PIF+PhyB:VP16; ?1Gal4:PIF+PhyB:VP16; ?1
1 µl LexA:PIF1 µl LacI:PIF1 µl Gal4:PIF
1 µl PhyB:VP16 (88E)1 µl PhyB:VP161 µl PhyB:VP16
1 µl ?11 µl ?11 µl ?1
1.2 µl buffer ligase1.2 µl buffer ligase1.2 µl buffer ligase
1.2 µl BSA1.2 µl BSA1.2 µl BSA
1 µl BsmbI1 µl BsmbI1 µl BsmbI
1 µl BsaI1 µl BsaI1 µl BsaI
4.6 µl H2O4.6 µl H2O4.6 µl H2O
35S:Bxb1+RepBxb1:GFP; ?1E-PIF+phyB+luc+ren; ?1
1 µl 35s:Bxb1:T35S (alfredo’s)0.5 µl 896 (PIF+phy+luc)
1 µl PromsinATG:RepBxb1:GFP1 µl 160 (renilla)
1 µl ?11 µl ?1
1.2 µl buffer ligase1.2 µl buffer ligase
1.2 µl BSA1.2 µl BSA
1 µl BsmbI1 µl BsmbI
1 µl BsaI1 µl BsaI
4.6 µl H2O4.6 µl H2O

The samples that we sent to sequence have arrived:

  • 210.08-249: pUPD2, KDronpa C3------ok
  • 210.08-250: pUPD2, NDronpa C1------ok
  • 210.08-251: pUPD2, NDronpa C3------ok
  • 210.08-252: pUPD2, NDronpa C4------ok

The sequences of N-Dronpa have the desired mutation.

  • Transformation in E.Coli of the 8 ligations.
  • Put the transformation into plates, put at 37ºC.
  • Refresh the Agro’s cultures (Renilla and PIF+phy+luc):
  • Add in 5ml of LB, 5 µl of rifampicine, 5 µl kanamicine, 5 µl of culture.

4 July 2015

Make the 2nd refresh of the culture of Agrobacterium. Put 5ml of LB, 5 µl of rifampicine, 5 µl kanamicine, 5 µl of culture.

Make liquid cultures (4ml of LB and 4 µl of spectomicine) of the 8 colonies of E.Coli.

  • Red toggle (C1)
  • Gal4:Kdronpa+N-Dronpa (C1 and C2)
  • LexA:Kdonpa+N-Dronpa (C1 and C2)
  • LacI:Kdronpa+N-Dronpa (C1 and C2)
  • LexA:PIF+PhyB:VP16 (C1 and C2)
  • 35S:Bxb1+RepBxb1:GFP (C1 and C2)
  • This colonies didn’t grow: LacI:PIF+PhyB:VP16; Gal4:PIF+PhyB:VP16 and E-PIF+phyB+luc+ren. Tomorrow we will repeat the ligations.

5 July 2015

Ligations:

LacI:PIF+PhyB:VP16; ?1Gal4:PIF+PhyB:VP16; ?1
1 µl LacI:PIF1 µl Gal4:PIF
1 µl PhyB:VP161 µl PhyB:VP16
1 µl ?11 µl ?1
1.2 µl buffer ligase1.2 µl buffer ligase
1.2 µl BSA1.2 µl BSA
1 µl BsmbI1 µl BsmbI
1 µl T4 ligase1 µl T4 ligase
4.6µl H2O4.6 µl H2O

Minipreps of the liquid cultures.

Digestion of the minipreps.

LacI:Kdronpa+N-DronpaBamHI6674, 5437
35S:Bxb1+RepBxb1:GFPBamHI6674, 3859, 1782
Gal4:Kdronpa+N-DronpaBamHI6674, 4666
LexA:Kdonpa+N-DronpaBamHI6674, 4705
LexA:PIF+PhyB:VP16BamHI6674, 3513, 2337
  • Agarose gel (1%):

LacKN C1 LacIKN C2 Bxb1RepGFP C1 Bxb1RepGFP C2 Gal4KN C1 Gal4KN C2

ok ok ok ok ok ok

LexA:KN C1 LexA:KN C2 LexAPIFPhy C1 LexAPIFPhy C2 Red toggle

ok ok no no no

We have to repeat the digestion of: LexA+PIF:phy+VP16.

  • Take out the glycerinate 88C (1098): Etr8:luc:Tnos. We will use it like a negative control in the second luciferase essat.
  • Calculation of the ODs:
    • Dilution of both samples 1:10.
    • Renilla:=0.22---182 µl of sample + 1.818 ml MES
    • PIF+PhyB+Luc=0.26--- 154 µl of sample + 1.646 ml MES

Agroinfiltration of the two samples with renilla and luciferase+PIF+phy in three plants with 4 spots in each leaf and 2 leaf in each plant.

Let the plants 2 days in the darkness till agrobacterium infects the plant. They have to be in the dark because we are trying our red toggle ant it activates with light.


6 July 2015

Transform the negative control into agrobacterium.

  • Etr8:luc:Tnos
  • Bxb1:reporterBxb1:GFP

We were doing dry lab preparing the power point to present our project to the rector and biotecs companies.


7 July 2015

Luciferase essay: Copy the protocol.

  • Add 150 µl of the lisis buffer and 800µl of MiliQ water, dilution 1:5.
  • We centrifuge both cultures of agrobacterium at 2900rpm for 10min and remove the supernatant.
  • We prepare the stock of MES (10ml of MES + 1ml MgCl + 100µl of “Acetosiningona” and level with H2O until 100ml.
  • Resuspend the pellet of bacteria with 5ml of MES and let grow 2h.
  • Renilla=0.29 (dilution 1:10): 2.9
  • PhyB+PIF+luc=0.69 (dilution 1:4):2.76
  • Solution to agroinflitrate:
  • Renilla: 0.138ml of sample + 1.862ml of MES
  • PhyB+PIF+luc: 0.145ml of sample + 1.855ml of MES
  • We make the infiltration of both samples mix together in 3 different plants, 2 leaf per plant and 4 spots per leaf. Explicacion del experiment (tiempo en oscuridad… luces…)

Digestions:

Red toggleEnzyme?
LexA+PIF+phyBamHI3518, 5855, 6674

Gel:

Red toggleLexA+PIF+phy C1LexA+PIF+phy C2
NoOkno

It has arrived a new construction: AsLOVpep.

  • Suspended with 50µl of H2O.

Ligations:

AsLOVpep; pUPD2Red ToggleLacI:Kdronpa:Ndronpa:VP16+
35S:renilla:Tnos-35S:P19:Tnos(GB159)Gal4:Kdronpa:Ndronpa:VP16+GB159
1 µl AsLOVpep1 µl GB8461 µl LacI:KNdronpa:VP161 µl Gal4:KNdronpa
1 µl pUPD21 µl GB1601 µl GB1591 µl GB159
1.2 µl buffer1 µl ?11 µl a11 µl a1
1.2 µl BSA1.2 µl buffer1.2 µl buffer1.2 µl buffer
1 µl BsmbI1.2 µl BSA1.2 µl BSA1.2 µl BSA
1 µl T4 ligase1 µl BsmbI1 µl BsmbI1 µl BsmbI
5.6 µl H2O1 µl T4 ligase1 µl T4 ligase1 µl T4 ligase
4.6 µl H2O4.6 µl H2O4.6 µl H2O
LexA:Kdronpa:Ndronpa:VP16+GB159LexA:PIF:Phy:VP16+ GB159
1 µl LexA:Kdronpa:Ndronpa:VP161 µl LexA:PIF:Phy:VP16
1 µl GB1591 µl GB159
1 µl a11 µl a1
1.2 µl buffer1.2 µl buffer
1.2 µl BSA1.2 µl BSA
1 µl BsmbI1 µl BsmbI
1 µl T4 ligase1 µl T4 ligase
4.6 µl H2O4.6 µl H2O

8 July 2015

  • Experiment to study the piece PhyB+PIF.

The plants in red are exposed at the light intensity of the leds (we can’t regulate it) and the plants in far red are 100% with far red light and 0% of white light.

How the machines work: (hace falta?)

The machine with red light has the switch …

The 1st sample its at 8:00am and we spend 1h till the machines were working correctly because we didn’t know exactly how they work.

Then, before 12h (21:00), we take the 2nd samples; obtainin 3 discs of each condition (red an far red).

We transform:

  • AsLOVpep; pUPD2 in DHSa an let incubate 1h at 37ºC.
  • The last ligations in E.coli. Put in plates. Red toggle with Spectomicine+IPTG+XGal ann the rest (a1) with kanamicine+IPTG+XGal.

9 July 2015

Pick colonies:

  • AsLOVpep colonies didn’t grow. Repeat.
  • Red toggle colonies are all blue. Repeat.
  • The other 4 colonies had grown. we pick them and male liquid culture.
  • LacI:Kdronpa:Ndronpa:VP16+renilla (C1-C3)
  • Gal4:Kdronpa:Ndronpa:VP16+renilla (C1-C3)
  • LexA:Kdronpa:Ndronpa:VP16+GB159 (C1 and C2)
  • LexA:PIF:Phy:VP16+ GB159 (C1 and C2)

Repeat the ligations.

  • AsLOVpep, as before.

Red toggle (PIF+PhyB+luc+ren)

0.5 µl PIF+phy+luc (896)

1 µl renilla (160)

1 µl ?1

1.2 µl buffer

1.2 µl BSA

1 µl BsmbI

1 µl T4 ligase

5.1 µl H2O

We do the luciferase essay:

  • We didn’t obtain goo results, we can’t observed a significative difference between the on(red samples) and off (far red samples). It seems that the red toggle it has been activated. Graphics and tables

Transform the ligations of AsLOVpepe and red toggle.

  • Add SOC medium and let incubate 1h at 37ºC. Then we make an spin to the red toggle cells to concentrate them and see if we can obtain a colonies in the plates.

10 July 2015

Minipreps of:

  • LexABD:PIF:phyB:VP16+Renilla; a1 (C1,C2)
  • LexA:Kdronpa:Ndronpa+renilla; a1C1, C2)
  • Gal4:Kdronpa:Ndronpa+renilla; a1 (C1-C3)
  • LacI:Kdronpa:Ndronpa+renilla; a1 (C1,C2)

Digestions of:

LexABD:PIF:phyB:VP16+Renilla; a1 (C1,C2)EcoRI6345, 5487, 4891
LexA:Kdronpa:Ndronpa+renilla; a1C1, C2)EcoRI9333, 6345
Gal4:Kdronpa:Ndronpa+renilla; a1 (C1-C3)EcoRI6345, 9194
LacI:Kdronpa:Ndronpa+renilla; a1 (C1,C2)EcoRI6345, 9965
LacIBD+PIF+phyB; Ω1 (C1)BamHI6674, 4245, 2337
Gal4BD+PIF+phyB; Ω 1 (C1)BamHI6674, 3474, 2337

Make the gel:

LacIBD+PIF+phyBGal4BD+PIF+phyBLexA:PIF:phyB:VP16+Renilla C1LexA:PIF:phyB:VP16+Renilla C2
OkOkokok
LexA:KNdronpa+renilla C1LexA:KNdronpa+renilla C2Gal4:KNdronpa+renilla C1Gal4:KNdronpa+renilla C2
OkOkokOk
Gal4:KNdronpa+renilla C3LacI:Kdronpa:Ndronpa+renilla C1LacI:Kdronpa:Ndronpa+renilla C2
OkOkok

We received two constructions:

  • CDS: phiC31. Resuspended with 100µl.
  • Reporter phi31. Resuspended with 50 µl

Pick colonies and make liquid culture of:

  • AsLOVpep; pUPD2 (C1 and C2)
  • Red toggle (C1 and C2). In both liquid cultures we ad YPTG and Xgal to make sure that the colonies are correct, if not the medium will change to blue color.

Ligations:

phyC31;pUPD2Reporter phiC31; pUPD2LacI:PIF:Phy:VP16+ren; a1
1 µl phiC311 µl rep phiC311 µl LacI:PIF:Phy:VP16
1 pUPD21 pUPD21 µl renilla (159)
1.2 µl buffer1.2 µl buffer1.2 µl buffer
1.2 µl BSA1.2 µl BSA1.2 µl BSA
1 µl T4 ligase1 µl T4 ligase1 µl T4 ligase
1 µl BsmbI1 µl BsmbI1 µl BSAI
5.6 µl H2O5.6 µl H2O4.6 µl H2O
1 µl a1
Gal4:PIF:phy:VP16+ren; a1LexA:PIF:phy:ren+opLex:luc; ?1LexA:KNdronpa:ren+OpLex:Luc; ?1
1 µl Gal4:PIF:phy:VP161 µl LexA:PIF:phy:ren1 µl LexA:KNdronpa:ren
1 µl renilla (159)1 µl opLex:luc (151)1 µl OpLex:Luc (151)
1.2 µl buffer1.2 µl buffer1.2 µl buffer
1.2 µl BSA 1.2 µl BSA1.2 µl BSA
1 µl T4 ligase1 µl T4 ligase1 µl T4 ligase
1 µl BSAI1 µl BsmbI1 µl BsmbI
4.6 µl H2O4.6 µl H2O4.6 µl H2O
1 µl a11 µl ?11 µl ?1
Gal4:KNdronpa:ren+UAS:luc; ?1LacI:KNdronpa:ren+OpLacI:luc; ?1
1 µl Gal4:KNdronpa:ren1 µl LacI:KNdronpa:ren
1 µl UAS:luc (227)1 µl OpLacI:luc (152)
1.2 µl buffer1.2 µl buffer
1.2 µl BSA 1.2 µl BSA
1 µl T4 ligase1 µl T4 ligase
1 µl BsmbI1 µl BsmbI
4.6 µl H2O4.6 µl H2O
1 µl ?11 µl ?1

11 July 2015

Prepare glycerinates of:

  • Bxb1+Rep:GFP; ?1
  • N-Dronpa; pUPD2
  • Bxb1:Etr8; pUPD2
  • Etr8(CMV):Bxb1:T35S; a1

Pick up the liquid cultures of AsLOVpep and red toggle. We observed that one tube of a red toggle colony is blue, we discard it.

Do miniprep of:

  • AsLOVpep (C1 and C2)
  • Red toggle (C2)

Digestions:

Red toggleBamHI6674, 6100 / 4209, 3756
AsLOVpepNotI2558, 512

Me falta el resultado del gel!!

AsLOVpep C1AsLOVpep C2Red toggle C2
¿?

Do transformation of DHSa and yesterday ligations:

  • phyC31;pUPD2
  • Reporter phiC31; pUPD2
  • LacI:PIF:Phy:VP16+ren; a1
  • Gal4:PIF:phy:VP16+ren; a1
  • LexA:PIF:phy:ren+opLex:luc; ?1
  • LexA:KNdronpa:ren+OpLex:Luc; ?1
  • Gal4:KNdronpa:ren+UAS:luc; ?1
  • LacI:KNdronpa:ren+OpLacI:luc; ?1
  • The pUPD2 in plates with cloranfenicol+IPTG+XGal. The a1 in plates with kanamicyn+IPTG+XGal. The ?1 in plates with spectinmicyn+IPTG+XGal.

12 July 2015

Pick colonies and make liquid culture:

  • phyC31;pUPD2 (C1-C3)
  • Reporter phiC31; pUPD2 (C1-C3)
  • LacI:PIF:Phy:VP16+ren; a1 (C1-C3)
  • Gal4:PIF:phy:VP16+ren; a1 All blue colonies.
  • LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
  • LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
  • Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
  • LacI:KNdronpa:ren+OpLacI:luc; ?1 All blue colonies.
  • We have to repeat the transformations or do again ligations.

Ligations were repeated:

Gal4:PIF:phy:VP16+ren; a1LacI:KNdronpa:ren+OpLacI:luc; ?1
1 µl Gal4:PIF:phy:VP161 µl LacI:KNdronpa:ren
1 µl renilla (159)1 µl OpLacI:luc (152)
1.2 µl buffer1.2 µl buffer
1.2 µl BSA 1.2 µl BSA
1 µl T4 ligase1 µl T4 ligase
1 µl BSAI1 µl BsmbI
4.6 µl H2O4.6 µl H2O
1 µl a11 µl ?1

Refresh the liquid cultures of Agrobacterium:

  • Bxb1:GFP and Etr8:tnos. They were 2 days at 28ºC.
  • Pnos, it was at the fridge (-4ºC).

13 July 2015

All the liquid cultures have grown, do minipreps.

Do digestions:

phyC31;pUPD2 (C1-C3)NotI2046, 1899
Reporter phiC31; pUPD2 (C1-C3)NotI2046, 475
LacI:PIF:Phy:VP16+ren; a1 (C1-C3)
EcoRI6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
BamHI9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
BamHI1199, 6674
Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
BamHI11582, 6674

Agarose gel:

phyC31 C1phyC31 C2phyC31 C3RepPhiC31 C1
nononook
RepPhiC31 C2 LacI:PIF:Phy:VP16+ren C1LacI:PIF:Phy:VP16+ren C2LacI:PIF:Phy:VP16+ren C3
nonookok
LexA:PIF:phy:ren+opLex:lucLexA:KNdronpa:ren+OpLex:Luc C1LexA:KNdronpa:ren+OpLex:Luc C2LexA:KNdronpa:ren+OpLex:Luc C3
nookokok
Gal4:KNdronpa:ren+UAS:luc C1
no

The Agrobacterium cultures refreshed yesterday were store in the fridge.

It is made another culture of 35S:Bxb1+reporterBxb1:GFP to keep it in the fridge. It had 5ml of LB medium, 5 µl rifampicin and 5 µl of spectinomicyn an 1 µl of the culture.

Mesurement of the OD’s:

  • 35S:Bxb1+reporterBxb1:GFP: 0.28 (dilution 1:10)
  • 143 µl of culture+1857 µl of MES/acetosiningon solution.
  • With this preparation 2 plants were infiltrated and let in natural light to see the normal activity of the recombinase.

Transformation of the ligation: Gal4:PIF:phy:VP16+ren; a1 and LacI:KNdronpa:ren+OpLacI:luc; ?1.

The liquid cultures of this constructions were repeated:

  • phyC31;pUPD2 (C4 and C5)
  • Reporter phiC31; pUPD2 (C4)
  • LacI:PIF:Phy:VP16+ren; a1 (C4)
  • LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
  • LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
  • Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
  • AsLOVpep; pUPD2 (C3)

14 July 2015

Do minipreps of:

  • phyC31;pUPD2 (C4 and C5)
  • Reporter phiC31; pUPD2 (C4)
  • LacI:PIF:Phy:VP16+ren; a1 (C4)
  • LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
  • LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
  • Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
  • AsLOVpep; pUPD2 (C3)

Do digestions:

phyC31;pUPD2NotI2046, 1899
Reporter phiC31; pUPD2NotI2046, 475
LacI:PIF:Phy:VP16+ren; a1EcoRI6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc, ?1BamHI9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; ?1BamHI1199, 6674
Gal4:KNdronpa:ren+UAS:luc; ?1BamHI11582, 6674
AsLOVpep; pUPD2 NotI2558, 512

Make an agarose gel:

phyC31 (C4)phyC31 (C5)AsLOVpep (C4)LexA:PIF:phy:ren+opLex:luc (C1)
nonoNoNo
LexA:KNdronpa:ren+OpLex:Luc (C3)Gal4:KNdronpa:ren+UAS:luc (C1)Gal4:KNdronpa:ren+UAS:luc(C2)Gal4:KNdronpa:ren+UAS:luc (C3)
OknonoNo
LacI:PIF:Phy:VP16+renReporter phiC31 (C1)
noOk

Only LexA:KNdronpa:ren+OpLex:Luc and Reporter phiC31 were correct. Repeat the ligations because is the second digestion of this construction that were made.

  • Measurement of DNA concentration:
    • Reporter:phyC31: 13.6 ng/µl
    • PhyC31: 4.6 ng/µl
    • AsLOVpep: 30.3 ng/µl
    • Igem151 (op:LexAluc): 40 ng/µl
    • Gal4:PIF:phyB:ren (C1): 124.4 ng/µl
    • LacI:PIF:phyB:VP16 (C1): 126 ng/µl
    • Igem 159 (renilla): 42 ng/µl
    • Gal4:Kdronpa:Ndronpa:renilla (C3): 172.8 ng/µl
    • Igem 227 (op:UAS:luc): 6.3 ng/µl
  • Pick colonies and make liquid culture of LacI:KDronpa:NDronpa:ren:luc (C4 and C5). The colonies of Gal4:PIF:phy:VP16+ren were all blue.

15 July 2015

  • Miniprep of LacI:KDronpa:NDronpa:ren:luc (C4 and C5).
  • Digestion:

LacI:KDronpa:NDronpa:ren:luc EcoRI 6345, 9965

  • Make the gel:

LacI:K:NDronpa:ren:luc C4 LacI:K:NDronpa:ren:luc C5

no no

  • Measurement of DNA concentrations:
    • Gal4BD:PIF:PhyB:VP16: 125.6 ng/µl
    • LexA:PIF:phyB:VP16:ren: 322.6 ng/µl
    • LacI:Kdronpa:NDronpa:ran: 209.0 ng/µl
  • We decided to repeat the ligations due to that we make twice the digestions and we didn’t obtain good results.

phyC31;pUPD2 AsLOVpep; pUPD2 LacI:PIF:Phy:VP16+ren; a1

1 µl phiC31 1 µl AsLOVpep 1.5 µl LacI:PIF:Phy:VP16

1 pUPD2 1 pUPD2 2.5 µl renilla (159)

1.2 µl buffer 1.2 µl buffer 1.2 µl buffer

1.2 µl BSA 1.2 µl BSA 1.2 µl BSA

1 µl T4 ligase 1 µl T4 ligase 1 µl T4 ligase

1 µl BsmbI 1 µl BsmbI 1 µl BSAI

5.6 µl H2O 5.6 µl H2O 4.6 µl H2O

0.5 µl a1

Gal4:PIF:phy:VP16+ren; a1 LexA:PIF:phy:ren+opLex:luc; ?1 LacI:KNdronpa:ren+OpLex:Luc; ?1

1.5 µl Gal4:PIF:phy:VP16 1 µl LexA:PIF:phy:ren 1 µl LacI:KNdronpa:ren

2 µl renilla (159) 2.5 µl opLex:luc (151) 3 µl OpLac:Luc (152)

1.2 µl buffer 1.2 µl buffer 1.2 µl buffer

1.2 µl BSA 1.2 µl BSA 1.2 µl BSA

1 µl T4 ligase 1 µl T4 ligase 1 µl T4 ligase

1 µl BSAI 1 µl BsmbI 1 µl BsmbI

2.6 µl H2O 2.6 µl H2O 3 µl H2O

1 µl a1 0.5 µl ?1 0.5 µl ?1

Gal4:KNdronpa:ren+OpLex:Luc; ?1 PhyB:VP16+PIF6; ?1

1 µl Gal4:KNdronpa:ren 1 µl PhyB:VP16 (88E)

4 µl OpUAS:Luc (227) 2 µl PIF6 (170)

1.2 µl buffer 1.2 µl buffer

1.2 µl BSA 1.2 µl BSA

1 µl T4 ligase 1 µl T4 ligase

1 µl BsmbI 1 µl BsmbI

3 µl H2O 3.6 µl H2O

0.5 µl ?1 1 µl ?1

  • These Agrobacterium cultures have been refreshed:
    • PIF-phyB-luc
    • Renilla
    • Pnos
    • Etr8

16 July 2015

  • Yesterday ligations have been transformed into E. coli:
    • phyC31;pUPD2
    • AsLOVpep; pUPD2
    • LacI:PIF:Phy:VP16+ren; a1
    • Gal4:PIF:phy:VP16+ren; a1
    • LexA:PIF:phy:ren+opLex:luc; ?1
    • LacI:KNdronpa:ren+OpLex:Luc; ?1
    • Gal4:KNdronpa:ren+OpLex:Luc; ?1
    • PhyB:VP16+PIF6; ?1
  • The agroinfiltrated leaf with BxbI:rep:GFP has been observed in the magnifying glass with fluorescent lights. The efficiency of the recombinase is lower and it can not been observed a lot of green spots. Tomorrow another leaf will be seen to check again the construction.
  • Second refresh of the Agrobacterium cultures:
    • PIF-phyB-luc
    • Renilla
    • Pnos
    • Etr8
  • Miniprep of these cultures.
  • Digestion of the minipreps:

PIF-phyB-luc; ?1 EcoRI ??

Renilla; ?2 HindIII No lo encuentro

Pnos; ?1 EcoRI 2997, 353

Etr8; ?1 EcoRI

The digestions had positive controls that were included in the gel to compare the results obtained.

The digestions are left overnight in the working table.


17 July 2015

Do the gel:

No se lo que pone?
  • Liquid culture of the colonies in the agar plates have been made, 3 colonies for each construction.
  • OD’s mesurement for the agroinfiltration.
    • Dilution 1:10 in MES.

PIF:phyB:luc 0.47 41 µl/ml 630 µl

Renilla 0.28 71 µl/ml 1065 µl

Etr8:luc 0.32 52 µl/ml 930 µl

Pnos 0.34 59 µl/ml 885 µl

  • Red toggle experiement:

The plants were coinfiltrated with the red toggle (PIF+phyB) and renilla. Also with two controls, Pnos (positive control) and Etr8 (negative control).

14 plants were used with 3 infiltrated leafs for each plants and two spots per leaf.

The controls have been infiltrated in leafs of the plants like is shown in the picture. Pnos in the right part and Etr8 in the left part.

Two plants were infiltrated with both controls in the three leafs and they were in natural light during all the experiment.

Another 4 plants were infiltrated with controls following the same pattern in the procedure.

The remaining 8 plants were infiltrated with the red toggle.

Immediately after the infiltration the plants were distributed in three different conditions: natural light, darkness and far red.

So after the agroinfiltration 2 control plants stay in natural light all the experiment; 2 control plants and 4 red toggle went into the far red chamber and the same amount of control and red toggle plan (2+4) went put in darkness.

This conditions were maintained 3 days and then all the infiltrated leafs were cut into small discs and put into a special plates with water.

In this moment was set the time 0 (21/07/2015 at 19:30) and take the first samples.

Then in time 0 some of the dark and far red samples were put in red conditions to activate the red toggle.

This scheme represent the distribution of samples and the times that were taken the samples.

Hacer el esquema!!! Cuando este mas espabilada ;)

It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights. It was not observed a lot of spots, the efficiency is very low.


18 July 2015

23 minipreps of the liquid cultrures. LexA:PIF:phtB:ren:luc (C3) has not grown.

Digestions of the minipreps:

phyC31;pUPD2NotI2046, 1899
AsLOVpep; pUPD2NotI
2046, 521
LacI:PIF:Phy:VP16+ren; a1EcoRI6345, 5623, 5487
Gal4:PIF:phy:VP16+ren; a1EcoRI6345, 5487, 4852
LexA:PIF:phy:ren+opLex:luc; ?1BamHI9431, 6674, 3513
LacI:KNdronpa:ren+OpLex:Luc; ?1BamHI12632, 6574
Gal4:KNdronpa:ren+OpLex:Luc; ?1BamHI11582, 6674
PhyB:VP16+PIF6; ?1BamHI6674, 2685, 2337, 1439

Gel has been done:

phyC31 C1phyC31 C2phyC31 C3AsLOVpep C1
nononook
AsLOVpep C2AsLOVpep C3Gal4:PIF:phy:VP16:ren C1Gal4:PIF:phy:VP16:ren C2
nononono
Gal4:PIF:phy:VP16:ren C3LacI:PIF:phy:ren C1LacI:PIF:phy:ren C2LacI:PIF:phy:ren C3
nooknoNo
LexA:PIF:phy:ren:luc C1LexA:PIF:phy:ren:luc C2Gal4:KNdronpa:ren:luc C1Gal4:KNdronpa:ren:luc C2
nonookNo
Gal4:KNdronpa:ren:luc C3LacI:KNdronpa:ren:luc C1LacI:KNdronpa:ren:luc C2LacI:KNdronpa:ren:luc C3
nonookNo
PhyB:VP16:PIF6 C1PhyB:VP16:PIF6 C2PhyB:VP16:PIF6 C3
oknono

Pick more colonies of:

  • Gal4:PIF:phy:VP16+ren; a1
  • LexA:PIF:phy:ren+opLex:luc; ?1

New digestions with new enzymes:

phyC31;pUPD2XhoI (buffer red)2119, 934, 894
LacI:PIF:Phy:VP16+ren; a1NEB45949, 5653, 3610, 2246
LacI:PIF:Phy:VP16+ren; a1HindIII11568, 5587

After 3 digestions of LacI:PIF:Phy:VP16+ren; a1 is accepted the construction.

It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights. It was not observed a lot of spots, the efficiency is very low. This is the 4th day…


19 July 2015


20 July 2015


21 July 2015

  • Red toggle experiment:

Time lapses:

-19:00=t0

-1:00=t1

-7:00= t2

-19:00= t3 (it was taken the controls in natural light)

  • Minipreps of the colonies that were in 37ºC.
    • LexA:PIF:Phy:ren:luc (C1 and C2)
    • PhyC31 (C1, C2, C4 and C5)
LexA:PIF:Phy:ren:lucBamHI9431, 6674, 3513
PhyC31NotI2046, 1899

Gel with the digestions:

PhyC31 C1PhyC31 C2PhyC31 C4PhyC31 C5
Ok?okokok
LexA:PIF:Phy:ren:luc C1LexA:PIF:Phy:ren:luc C2
No DNANo DNA

We had problems with some colonies because in the digestion did not appear DNA. The minipreps will be made with a better kit.

Transform in Agrobacterium this cultures:

  • LexABD:KDronpa:NDronpa:ren:luc
  • Gal4BD:KDronpa:NDronpa:ren:luc
  • LacIBD:KDronpa:NDronpa:ren:luc
  • OpLexA:luc (151)
  • OpUAS:luc
  • OpLacI:luc (152)

It was made liquid culture of Gal4:PIF:phyB:ren; ?1 (C1-C5)

It was made ligations:

35S:Gal4:AsLOVpep:T35S; ?135S:LacI:AsLOVpep:T35S; ?135S:LexA:AsLOVpep:T35S; ?1
1 µl 35S (0030)1 µl 35S (0030)1 µl 35S (0030)
1 µl Gal4BD1 µl LacI1 µl LexA
1 µl AsLOVpep 1 µl AsLOVpep 1 µl AsLOVpep
1 µl T35S (0036)1 µl T35S (0036)1 µl T35S (0036)
1 µl ?1 1 µl ?1 1 µl ?1
2.6 µl H2O2.6 µl H2O2.6 µl H2O
PsinATG:RepPhiC31:GFP:T35S; ?2LacI:PIF:PhyB:ren+luc; ?1PIF:PhyB+renilla; ?2
1 µl PsinATG (552)1.5 µl LacI:PIF:PhyB:ren; ?11.5 µl PIF:PhyB
1 µl ReporterPhyC313 µl OpLacI:luc (152); ?22.5 µl renilla (159)
1 µl GFP (0059)0.5 µl ?10.5 µl ?2
1 µl T35S (0036)2.6 H2O3 µl H2O
1 µl ?2
2.6 µl H2O

Luciferase essay with all the samples collected in the last three days.

  • Sampling: 25 samples in total.
  • Passive lysis 1x (200µl/sample x 25 samples)= 5.000 µl
  • Crush samples.
  • Add 150 µl of passive lysis buffer and mix in the vortex.
  • Centrifuge at 13200rpm for 15’.
E8/liE8/liE8/liP/liP/liP/liTR/FrTR/FrTR/FrTR/DTR/DTR/D
TR/Fr
RedTR/Fr
RedTR/r
RedTR/D
RedTR/D
RedTR/D
Red

We used 14.4 µl of Stop and Glow. 705.6 destilled H2O


22 July 2015

Miniprep of Gal4:PIF:PhyB:ren (C1-C3) C4 and C5 did not grow.

Digestion of the miniprep:

Gal4:PIF:PhyB:renBamHI16684
EcoRI4852, 5487, 6345
EcoRV1849, 3942, 2475, 381, 8037

Gel was made:

Gal4… (BamHI)Gal4… (EcoRI)Gal4… (EcoRV)
nonono

After doing several digestions with different enzyme all with wrong band patterns, it was decided to revise each part making digestions. The parts are:

  • PhyC31; pUPD2
  • Gal4:PIF:phyB;
  • Renilla (GB159)

Made liquid culture of the ReporterBxbI:GFP in Agrobacterium.

Transform the ligations into E. coli:

  • 35S:Gal4:AsLOVpep:T35S; ?1
  • 35S:LacI:AsLOVpep:T35S; ?1
  • 35S:LexA:AsLOVpep:T35S; ?1
  • PsinATG:RepPhiC31:GFP:T35S; ?2
  • LexA:PIF:PhyB:ren+luc; ?1
  • Add 300 µl of SOC medium and put into a agar plate with the corresponding antibiotics.

Luciferase essay:

  • Sampling: samples that have been in red and natural light 48h, 3 samples.
  • 200 µl/sample x 3 sample= 600 µl of passive lissis buffer (5x)
  • Passive lissis buffer 1x= 120 µl+480 µl water.
  • 2.4 µl of Stop and glow
  • 118 µl of buffer.

23 July 2015

We decided to infiltrate soybean sprouts. First we decided to infiltrate with dye to observe the characteristics and the capacity of absorption.

Mesurement of the ODs to agroinfiltrate:

The samples are:

  • Citoplasm=0.27
  • DsRed=0.27
  • GFP=0.36
  • Recombinase=0.43

Vi= (Vf*Absf)/(Absi*10)

Absi=0.1 (because is a viral system)

Vf=120 µl

Make 16 liquid culture of the white colonies in the agar plates.


24 July 2015

Minipreps of the 13 liquid culture. LacI:PIF:phyB:ren cultures did not grow.

Digestion of the minipreps:

Gal4:AsLOVpepEcoRI6345, 1972
LacI:AsLOVpepEcoRI6345, 2743
LexA:AsLOVpepEcoRI6345, 2011
PsinATG:RepPhiC31:GFPHindIII6345, 2691
LexA:PIF:PhyB:ren+lucBamHI9431, 6674, 3513
PhyC31; pUPD2NotI2046, 1899
Gal4:PIF:phyBBamHI6674, 3474, 2337
Renilla (GB159)EcoRV2909, 2475, 882, 812, 381

It was done 2 gels with ligations:

Gal4:AsLOV C1Gal4:AsLOV C2Gal4:AsLOV C3PsinATG:RepPhi
C31:GFP C1PsinATG:RepPhi
C31:GFP C2PsinATG:RepPhi
C31:GFP C3
okokokoknoNo
Mw/ladderLacI:AsLOV C1LacI:AsLOV C2LexA:AsLOV C1LexA:AsLOV C2LexA:AsLOV C3
-okNonookno
LexA:PIF:PhyB:ren+luc C1LexA:PIF:PhyB:ren+luc C2
noOk
PhyC31 (C1)PhyC31 (C2)PhyC31 (C3)PhyC31 (C4)PhyC31 (C5)
nookokokno
Gal4:PIF:phyBRenilla (GB159)
okOk

26 July 2015

Liquid cultures of Agrobacterium were refreshed:

  • TsinATG:BxbIreporter:GFP
  • TsinATG:BxbI:reporterBxbI:GFP
  • Viral vector??no se cual es

27 July 2015

Sent to sequence:

  • 210.08.256: LacIBD; pUPD2 (C1)
  • 210.08258: Gal4BD; pUPD2 (C2)
  • 210.08.259:LexABD; pUPD2 (C1)
  • 210.08.260: PIF6; pUPD2 (C5)
  • 210.08.261: VP16; pUPD2 (C1)
  • 210.08.262: PhiC31; pUPD2 (C2)
  • 210.08.264: PhiC31; pUPD2 (C3)
  • 210.08.266: PhiC31; pUPD2 (C4)
  • 210.08.268: ReporterBxbI; pUPD2 (C1)
  • 210.08.269: ReporterPhiC31; pUPD2 (C1)
  • 210.08.270: AsLOVpep; pUPD2 (C1)

The sample had to have 10µl of miniprep (200ng/µl aprox) + 5 µl of primer (dilution 1:3)

Ligations:

Gal4:PIF:phiB + ren; ?1LacI:PIF:phiB:ren + luc; ?2PIF:PhyB+renilla; ?2
1.5 µl Gal4:PIF:phiB1.5 µl LacI:PIF:phiB:ren1.5 µl PIF:phiB
2 µl Renilla (GB159)2 µl OpLacI:luc2.5 µl Renilla (GB159)
0.5 µl ?10.5 µl ?20.5 µl ?2
3.6 µl H2O2.6 µl H2O3 µl H2O

OD’s measurements to prepare the agroinfiltrates:

Cytoplasm: 0.39 (viral)0.25ml
Integrase: 0.35 (viral)0.28ml
GFP: 0.32 (viral)0.31ml
Dsred: 0.31 (viral)0.32ml
BxbI:reporter: 0.410.49ml
Reporter: 0.260.77ml

3 plants were infiltrated with BxbI and the reporter of BxbI (one leaf with the control an the other with the recombinase)?? No entiendo lo de la libreta

Ligations to join the negative controls with renilla:

Etr8:luc+staffer (SF)OpLexA:luc+SFOpLacI:luc+SFUAS:luc+SF
1.5 µl Etr8:luc (88C o 1098)1.5 µl OpLexA:luc (151)1.5 µl OpLacI:luc (152)1.5 µl UAS:luc (227)
1 µl SF; ?21 µl SF; ?21 µl SF; ?21 µl SF; ?2
1 µl ?11 µl ?11 µl ?11 µl ?1
6.1 µl H2O6.1 µl H2O6.1 µl H2O6.1 µl H2O

Transformation of E. coli of the ligations:

  • Gal4:PIF:phiB + ren; ?1
  • LacI:PIF:phiB:ren + luc; ?2
  • PIF:PhyB+renilla; ?2

Make cultures in agar petri dishes of the transformations with the corresponding antibiotics.

Transformation into Agrobacterium with the constructions:

  • Gal4:KDronpa:NDronpa:ren:luc
  • LacI:KDronpa:NDronpa:ren:luc
  • LexA:KDronpa:NDronpa:ren:luc
  • OpLexA:luc (GB 151)
  • OpLacI:luc (GB 152)
  • UAS:luc (GB 227)

Make cultures in agar petri dishes of the transformations with the corresponding antibiotics.

It was received a new piece (ePDZ) which is part of the blue toggle (plan A). It arrived in E.coli so it was made a lquid culture and let it grow at 37ºC overnight.


28 July 2015

Miniprep of the liquid culture: ePDZ.

Transformation into E.coli of the ligations:

  • Etr8:luc+staffer (SF)
  • OpLexA:luc+SF
  • OpLacI:luc+SF
  • UAS:luc+SF
  • Gal4:PIF:phyB:ren

It was observed the soybean sprouts that were infiltrated with a dye with green light and red filter. It was not observed nothing significant, moreover, the damage is evident.

Transformation in Agrobacterium the ReporterPhiC31:GFP.

Pick colonies and make liquid cultures adding X-Gal and IPTG because the colonies were little and we can not observe clearly if they were white or blue.

  • Gal4:PIF:phiB + ren. Did not grow any colony.
  • LacI:PIF:phiB:ren + luc (C1-C3)
  • PIF:PhyB+renilla (C1- C3)

The medicine LTB (heat labile toxin B subunit) which is part of an enterotoxin of Echerichia coli homologous to the same toxin in Vibrio cholerae that causes diarrhea.

We add 50µl to have a final concentration of 10ng/µl.

Ligation:

LTB; pUPD2
1 µl LTB
1 µl pUPD2
5.6 µl H2O

29 July 2015

Miniprep of yesterday liquid culture:

  • LacI:PIF:phiB:ren + luc (C1 and C3) C2 turn into blue.
  • PIF:PhyB+renilla (C2) C1 and C3 did not grow.

Digestion:

LacI:PIF:phiB:ren:lucEcoRV882, 968, 1652, 3942, 2475, 381, 3477, 6674
PIF:PhyB+renillaHindIII4316, 5887, 788, 6345

Gel:

LacI:PIF:phiB:ren:luc (C1)LacI:PIF:phiB:ren:luc (C3)PIF:PhyB+renilla (C2)
nonono

Pick colonies and make liquid culture of:

  • Etr8:luc:staffer(SF) (C1-C3)
  • OpLexA:luc:SF (C1-C3)
  • OpLacI:luc:SF (C1-C3)
  • UAS:luc:SF (C1-C3)
  • Gal4:PIF:phyB:ren (C1-C3)

Transformation in E.coli of:

  • LTB; pUPD2

30 July 2015

Minipreps have been done:

  • Etr8:luc:staffer(SF) (C1-C3)
  • OpLexA:luc:SF (C1 and C3) C2 did not grow.
  • OpLacI:luc:SF (C1-C3)
  • UAS:luc:SF (C1-C3)
  • Gal4:PIF:phyB:ren (C1-C3)
  • LacI:PIF:phy:ren:luc (C1-C3)
  • PIF:phyB:ren (C1-C3)

Etr8:luc:staffer(SF) BamHI 6674, 2766

OpLexA:luc:SF BamHI 6674, 2746

OpLacI:luc:SF BamHI 6674, 2847

UAS:luc:SF BamHI 6674, 2568

Gal4:PIF:phyB:ren EcoRI 6345, 5487, 4852

LacI:PIF:phy:ren:luc BamHI 20451

PIF:phyB:ren HindIII 6345, 5887, 4316, 788

Do the gel:

No se el orden!! Preguntar

Primers have arrived:

  • ePDZ reverse and forward and phyC31.

Make a PCR with ePDZ and its primers to obtain the desired fragment and put it in pUPD2.

ePDZ PCR
10µl Buffer HF
31.5 µl H2O
2 µl dNTPs
2.5 µl Primer forward
2.5 µl primer reverse
1 µl ePDZ (dilution 1:50)
0.5 µl Taq phunion

Pick 2 colonies of phiC31:GFP in Agrobacterium and make liquid culture.

Ligations:

ePDZ; pUPD2PIF:phyB+ren; ?2OpUAS:luc:SF+ren; ?1OpLexA:luc:SF+ren; ?1
1 µl ePDZ1 µl PIF:phyB1 µl OpUAS:luc:SF1 µl OpLexA:luc:SF
1 µl pUPD21 µl ren (159)1 µl ren1 µl ren
5.6 µl H2O1 µl ?21 µl ?11 µl ?1
4.6 µl H2O4.6 µl H2O4.6 µl H2O
OpEtr8:luc:SF+ren; ?1Op:LacI:luc:SF+ren; ?1
1 µl OpEtr8:luc:SF1 µl OpLacI:luc:SF
1 µl ren1 µl ren
1 µl ?11 µl ?1
4.6 µl H2O4.6 µl H2O

After 3 days till the agroinfiltration we have observed the leaf discs that have BxbI+repBxbI and the repBxbI (negative control).

We could see that the level of GFP expression was higer in the infiltration with the recombinase but there was also expression in the negative control. We think that this can be a contamination due to that we had infiltrated both constructions in he same leaf and we did not change the gloves between agroinfiltrations. FOTOO!

Make liquid culture (E.coli) of:

  • LTB; pUPD2 (C1-C3)

31 July 2015

Ligation:

LacI:PIF:phyB:ren+OpLacI:luc; ?2
1.5 µl LacI:PIF:phyB:ren
3 µl OpLacI:luc
0.5 µl ?2
2.6 µl H2O

After 4 days till the agroinfiltration we have observed again the samples with BxbI and the negative control but was the same. The negative control expressed GFP but less than the recombinase. Foto!

Minipreps of liquid culture LTB (C1 and C2)

Digestion:

LTB; pUPD2NotI2046, 474

Gel:

LTB (C1)LTB (C2)PCR ePDZ
??

Take out glicerynates of Paloma, lab mate:

  • Sip rotavirus CH2
  • Sip rotavirus CH2-CH3

For E.coli and Agrobacterium.

Pick a colony of Asun interferon (IFN) in Agro, make liquid culture.

We had miniprep of IFN in pUPD. Make ligations.

Transform in E.coli the ligations and make petri dishes cultures:

  • ePDZ; pUPD2
  • PIF:phyB+ren; ?2
  • OpUAS:luc:SF+ren; ?1
  • OpLexA:luc:SF+ren; ?1
  • OpEtr8:luc:SF+ren; ?1
  • Op:LacI:luc:SF+ren; ?1
  • LacI:PIF:phyB:ren+OpLacI:luc; ?2

Refresh agro cultures to agroinfiltrate tomorrow:

  • PhiC31 (viral system)
  • ReporterPhiC31
  • BxbI+reporterBxbI
  • ReporterBxbI
  • Gal4:KDronpa:NDronpa:luc:ren (brue toggle)
  • PIF:phi:luc
  • Renilla
  • P19
  • Pnos

New experiment with Nicotiana bentamiana. PROTOCOL

Next constructions were agroinfiltrated, 2 or 3 leafs for each plant:

PhiC31: one leaf for PhiC31+RepPhiC31+P19 (coinfiltration) and the other with RepPhiC31+P19 which is the negative control. 2 plants were infiltrated.

BxbI: one leaf with BxbI:RepBxbI+P19 and the other with RepBxbI+P19 which is negative control. 2 plants were infiltrated.

Red toggle: PIF6:phyB:luc+ren (coinfiltration). They were infiltrated 3 plants with 3 leafs for each.

Blue toggle: Gal4:NDronpa:KDronpa:luc:ren. They were infiltrated 3 plants with 3 leafs for each.

Pnos, the positive control. There are 4 plantas, 3 leafs per plant.

So we will take samples in time 0, 12, 24 and 36h for each construction and control. After 2 days of the agroinfiltration (during this period all the plants were in dark, it is set time 0 we make discs os leaf and put in a plate with water and we change the conditions that were before.

Red toggle plants: 9 discs stay in dark, 9 went to red and 9 went to natural light.

Blue toggle: the same as red but went to ultraviolet instead of red light.

Pnos: one plant were in natural light during all the experiment. The other discs will go, after the 2 days in black, to red, ultraviolet and stay in dark.

Recombinases: they are in natural light during all the experiment.


1 August 2015

Prepare the Agrobacterium cultures for agroinfiltration:

Gal4B:KDronpa:NDronpa:luc:ren, PhiC31 and its control, BxbI and its control and PIF:PhyB and its control were centrifugated 10 min at 3000g. The sobrenadant was discarded and the bacterias were resuspended with the agroinfiltration medium.

Agroinfiltration medium had: 10ml MES, 1ml MgCl2, 89ml H2O, 100ml DMSO+acetosiningone.

Incubate in 2h.

Mesurement oof the ODs:

Ren+P190.09888.9 µl
RepPhiC310.69115.94 µl
BxbI0.46174 µl
RepBxbI0.15533.3 µl
Gal4:KNDronpa:ren:luc0.51156.9 µl
Pnos0.29275.9 µl
PIF:phy:luc0.17470.6 µl
PhiC310.32250 µl

We went to the greenhouse and infiltrate the 13 plants.

Look the petri dishes culture:

  • PIF:phyB+ren; ?2
  • OpLexA:luc:SF+ren; ?1
  • Op:LacI:luc:SF+ren; ?1

This colonies did not grow, the rest were left in the fridge.

  • ePDZ; pUPD2
  • OpUAS:luc:SF+ren; ?1
  • OpEtr8:luc:SF+ren; ?1
  • LacI:PIF:phyB:ren+OpLacI:luc; ?2

3 August 2015

Miniprep of the liquid culture:

  • Sip rotavirus CH2
  • Sip rotavirus CH2-CH3

Measure of DNA concentration of:

  • OpLexA:luc:SF=169ng/µl
  • OpacI:luc:SF=291ng/µl

Pick colonies gb159and make liquid culture of:

  • ePDZ; pUPD2 (C1-C3)
  • OpUAS:luc:SF+ren; ?1(C1-C3)
  • OpEtr8:luc:SF+ren; ?1(C1-C3)
  • LacI:PIF:phyB:ren+OpLacI:luc; ?2 (C1-C3) Added X-gal and IPTG.

Repeat ligations:

OpLexA:luc:SF+ren; ?1Op:LacI:luc:SF+ren; ?1E-PIF:phyB+ren; ?2
1 µl OpLexA:luc:SF1 µl OpLacI:luc:SF1 µl E-PIF:phy
3 µl ren3 µl ren3 µl ren
1 µl ?11 µl ?11 µl ?1
2.6 µl H2O2.6 µl H2O2.6 µl H2O

Refresh agrobacterium cultures of:

  • Interferon
  • SIP-CH2
  • SIP-CH2-CH3

Take the glycerinate of Renilla; ?2 (GB159) and make liquid culture.

We have made all the leaf discs and put in order in the plates with 300 µl of water, also we put each plate in the conditions light that they have to be. We have taken the samples of time0 (13:00).

So as T0= 13:00, T12=1:00, T24= 13:00 and T36= 1:00.

Imagenes de la colocacion en los platos? Hace falta?


4 August 2015

We do a Western blot with Asun so we can learn how to do it. Lo copio? De verdad!!?

Miniprep of the liquid cultures:

  • ePDZ; pUPD2 (C1-C3)
  • OpUAS:luc:SF+ren; ?1(C1-C3)
  • OpEtr8:luc:SF+ren; ?1(C1-C3)
  • LacI:PIF:phyB+ren; ?2 (C2 and C3) C1 was blue.

Digestions:

ePDZ; pUPD2NotI2046, 642
OpUAS:luc:SF+ren; ?1EcoRI6345, 7096
OpEtr8:luc:SF+ren; ?1EcoRI6345, 7294
LacI:PIF:phyB:ren+OpLacI:luc; ?2EcoRV6674, 3477, 381, 2475, 3942, 1652, 968, 882
Renilla 159EcoRV2909, 2475, 882, 812, 381

Gel:

ePDZ C1ePDZ C2ePDZ C3LacI:PIF:phyB+ren
Okokok
Renilla 159OpUAS:luc:SF+ren C1OpUAS:luc:SF+ren C2OpUAS:luc:SF+ren C3
Ok¿?
OpEtr8:luc:SF+ren C1OpEtr8:luc:SF+ren C2OpEtr8:luc:SF+ren C3

Transformation into E.coli of yesterday ligations:

  • OpLexA:luc:SF+ren; ?1
  • Op:LacI:luc:SF+ren; ?1
  • E-PIF:phyB+ren; ?2

Make petri dishes culture with the indicate antibiotic.

Ligations:

35S+ePDZ+VP16+T35S;?235S+LTB+T35S; ?1
1µl ePDZ1µl 35S (30)
1 µl VP161µl T35S (36)
1 µl 35S (30)1µl LTB
1 µl T35S (36)1µl ?1
1 µl ?23.6 µl H2O
2.6 µl H2O

Refresh agrobacterium cultures of:

  • Interferon
  • SIP-CH2
  • SIP-CH2-CH3

5 August 2015

Transform ligations:

  • 35S+ePDZ+VP16+T35S; ?2
  • 35S+LTB+T35S; ?1

Pick colonies and make liquid cultures cultures of:

  • OpLexA:luc:SF+ren; ?1 (C1-C3)
  • Op:LacI:luc:SF+ren; ?1 (C1-C3)
  • E-PIF:phyB+ren; ?2 (C1)
    • 35S+ePDZ+VP16+T35S; ?2 (C1 and C2)
  • 35S+LTB+T35S; ?1 (C1 and C2)

Make luciferase essay of red and blue toggle:

Number of samples=75 (a lot)

1. Prepare lisis buffer for 80 samples.

200 µl/sample * 80sample = 16ml

Buffer (5x)—3.2ml of buffer + 12.8ml H2O miliQ: lysis buffer (1x)

2. Crushed samples+ 150 µl of lysis buffer.

3. Centrifuge 15min at 13200rpm in cold.

4. Dilution 2:3 (Add 36 µl lysis buffer + 24 µl sample)

5. Luciferase: 40 µl/sample. Prepare 2.88ml (3 substrate tubes)

6. Renilla:….?


6 August 2015

Miniprep of the liquid cultures.

Digestion:

OpLexA:luc:SF+renEcoRI6345, 7274
Op:LacI:luc:SF+renEcoRI6345, 7375
E-PIF:phyB+renHindIII6345, 788, 5887, 4316
35S+ePDZ+VP16+T35SHindIII6345, 2316
35S+LTB+T35SEcoRI6345, 1684

Gel:

PIF:phyB+renOpLexA:luc:SF+ren C1OpLexA:luc:SF+ren C2OpLexA:luc:SF+ren C3
NoOk, repeatNoOk, repeat
Op:LacI:luc:SF+ren C1Op:LacI:luc:SF+ren C2Op:LacI:luc:SF+ren C335S+LTB+T35S C1
Ok, repeatOkOkok
35S+LTB+T35S C235S+ePDZ+VP16+T35S C135S+ePDZ+VP16+T35S C2
Okokok

Make colony PCR with 6 colonies of PhiC31:

  • DNA-
  • JM1: 2 µl (dilution 1:10)
  • JM2: 2 µl (dilution 1:10)
  • Buffer Taq (with Mg): 2 µl
  • dNTPs: 2.5 µl
  • Taq: 0.5 µl
  • H2O: 10 µl
  • Program: 96ºC-2min; 96ºC-30min; 55ºC-30min; 72ºC-30min

Make a gel with the PCR but we did not see the correct bands.

Repeat the colony PCR:

  • DNA-
  • JM1: 2.5 µl (dilution 1:10)
  • JM2: 2.5 µl (dilution 1:10)
  • Buffer Taq (with Mg): 10 µl
  • dNTPs: 2 µl
  • Taq: 0.5 µl
  • H2O: 7.5 µl
  • Program: : 96ºC-10min; 96ºC-30min; 55ºC-30min; 72ºC-30min

Ligations:

LexA:AsLOVpep+ePDZ; ?1LacI:AsLOVpep+ePDZ; ?1Gal4:AsLOVpep+ePDZ; ?1
1 µl LexA:AsLOVpep; ?11 µl LacI:AsLOVpep; ?11 µl Gal4:AsLOVpep; ?1
1 µl ePDZ; ?21 µl ePDZ; ?21 µl ePDZ; ?2
1 µl BsmBI1 µl BsmBI1 µl BsmBI
1 µl ?11 µl ?11 µl ?1
4.6 µl H2O4.6 µl H2O4.6 µl H2O

Refresh Agro cultures to agroinfiltrate:

  • PhiC31:RepPhiC31:GFP
  • RepPhiC31:GFP
  • BxbI:RepBxbI:GFP
  • RepBxbI:GFP
  • IFN (interferon)
  • Sip-CH2
  • Sip-CH2-CH3
  • Pnos
  • P19 (this culture ha 10ml of LB + 10 µl antibiotics + 5 µl culture)

7 August 2015

Transformation into Agrobacterium:

  • 35S:LTB:VP16:T35S

Transformation into E.coli and make petri dishes cultures:

  • LexA:AsLOVpep+ePDZ
  • LacI:AsLOVpep+ePDZ
  • Gal4:AsLOVpep+ePDZ

Make a gel with the colonies PCRs:

C7C8C9C10C11C12C13C14
Nononononononono

There was no DNA, there is a problem with the cells or the procedure, it have to be revised.

Ligation:

PhyC31; pUPD2
3 µl PhiC31
1 µl pUPD2
1 µl BsmBI
3.6 µl H2O

Refresh agrobacterium cultures for tomorrow infiltration:

  • Sip-CH2
  • Sip-CH2-CH3
  • Pnos
  • Red toggle (PIF6:PhyB:luc )
  • Renilla
  • Blue toggle (….:KDronpa:NDronpa:ren:luc)
  • A new experiment was started:
    • We are going to check again the recombinase BxbI and its reporter (negative control).
  • Test the recombinase PhiC31. Due to that we did not obtain yet the complete recombinase, we will coinfiltrate the PhiC31 and the RepPhiC31:GFP following the same scheme as BxbI. 2 plants with 2 leafs, one of them with PhiC31 + RepPhiC31:GFP and the other with only RepPhiC31:GFP.
  • Infiltration in 2 plants with 2 leafs each of them with interferon.

Measure f the ODs:

ConstructionODVolume (ml)
IFN0.131.54
RepBxbI:GFP0.131.54
BxbI:RepBxbI:GFP0.330.61
PhiC310.191.05
RepPhiC31:GFP0.220.91

8 August 2015

Transform the ligation into E.coli and make petri dishes cultures:

  • PhiC31; pUPD2.
  • LexA:AsLOVpep+ePDZ (C1-C5)
  • LacI:AsLOVpep+ePDZ (C1-C4)
  • Gal4:AsLOVpep+ePDZ (C1-C4)

New experiment:

It will be tested again the red toggle (PIF6:PhyB:luc + ren), the blue toggle (LacI:KDronpa:NDronpa:ren:luc), pnos (positive control to check the ratio renilla luciferasa) and the production of antirotavirus that are SIP rotavirus CH2 and SIP rotavirus CH2-CH3.

2 plants with 3 leafs ache one were infiltrated with pnos.

The same with the red(4) and blue toggle(4) and sip rativurs I DON’T KNOW

Measurement of the ODs:

ConstructionODVolume (ml)
Sip-CH20.046.667
Sip-CH2-CH30.046.667
Red toggle (PIF6:PhyB:luc)0.0915
Blue toggle (LacI:KDronpa:NDronpa:ren:luc)0.0915.00
Renilla0.046.667
Pnos0.046.667

9 August 2015

Make a colony PCRs with the 10 white colonies of PhiC31 that grow in the petri dishes.

  • DNA- colony
  • JM1: 2 µl (dilution 1:10)
  • JM2: 2 µl (dilution 1:10)
  • Buffer Taq (with Mg): 2 µl
  • dNTPs: 2.5 µl
  • Taq: 0.5 µl
  • H2O: 1 µl

Minipreps of the liquid cultures:

  • LexA:AsLOVpep+ePDZ (C1-C4) C5 has turn into blue color.
  • LacI:AsLOVpep+ePDZ (C1-C4)
  • Gal4:AsLOVpep+ePDZ (C1-C4)

Digestion of the minipreps:

LexA:AsLOVpep+ePDZBamHI6674, 4309
LacI:AsLOVpep+ePDZBamHI6674, 5041
Gal4:AsLOVpep+ePDZBamHI6674, 4270

Make the gel:

LexA:AsLOVpep+ePDZ (C1)LexA:AsLOVpep+ePDZ (C2)LexA:AsLOVpep+ePDZ (C3)LexA:AsLOVpep+ePDZ (C4)
Okokokok
oLacI:AsLOVpep+ePDZ (C1)LacI:AsLOVpep+ePDZ (C2)LacI:AsLOVpep+ePDZ (C3)LacI:AsLOVpep+ePDZ (C4)
Okokokok
Gal4:AsLOVpep+ePDZ (C1)Gal4:AsLOVpep+ePDZ (C2)Gal4:AsLOVpep+ePDZ (C3)Gal4:AsLOVpep+ePDZ (C4)
OkokokOk

We keep in our inventory the colony C1 of each construction

Make a gel of the colonies PCRs of PhiC31: we did not observed any DNA. Still can’t fix the problem.

Make liquid culture of renilla and PIF6:phyB:luc in Agrobacterium because the last time that we did an experiment they have grown slowly.

Store in the 4ºC fridge an agrobacterium culture with P19.

Ligations:

LexA:AsLOVpep+ePDZ+ren; ?1LacI:AsLOVpep+ePDZ+ren; ?1Gal4:AsLOVpep+ePDZ+ren; ?1
1µl LexA:AsLOVpep+ePDZ1µl LacI:AsLOVpep+ePDZ1µl Gal4:AsLOVpep+ePDZ
1 µl renilla (159)1 µl renilla (159)1 µl renilla (159)
1 µl ?11 µl ?11 µl ?1
4.6 µl H2O4.6 µl H2O4.6 µl H2O

10 August 2015

Transform into E.coli this ligations and make petri dishes cultures:

  • LexA:AsLOVpep+ePDZ+ren; ?1
  • LacI:AsLOVpep+ePDZ+ren; ?1
  • Gal4:AsLOVpep+ePDZ+ren; ?1
  • (we add into the tubes X-gal and IPTG)

Make liquid culture of 11 colonies of PhiC31 due to that this construction was giving us problems to obtain.

Refresh the agrobacterium cultures of:

  • BxbI:Rep:BxbI:GFP
  • Rep:BxbI:GFP
  • PhiC31
  • RepPhiC31:GFP
  • P19
  • Citoplasm
  • Integrase
  • Dsred
  • GFP

Take the samples of the agroinfiltrated plants that were in darkness for 2 days and make discs of them to change the conditions.

Make liquid culture of:

  • LexA:AsLOVpep+ePDZ+ren (C1 and C2)
  • LacI:AsLOVpep+ePDZ+ren (C1-C4)
  • Gal4:AsLOVpep+ePDZ+ren (C1 and C2)
  • (we add X-Gal and IPTG because they are small)

11 August 2015

Primers have arrived, they have been resuspended. They were used to eliminate the recognitions sites of the enzymes in BioBricks. This will make our constructions ready to be send to the iGEM

Minipreps of the liquid cultures done yesterday:

  • PhiC31 (C6-C16)
  • LacI:AsLOVpep+ePDZ+ren (C1-C4)
  • Gal4:AsLOVpep+ePDZ+ren (C1 and C2)
  • (LexA:AsLOVpep+ePDZ+rencolonies were all blue)

Make PCRs with all the primers and constructions:

All of them follow the same composition which is:

10 µl Buffer HF
26.5 µlH2O
2 µldNTPs
0.5 µlTaq Phusion
1 µl DNA (dilution 1:10)
2.5 µlPrimer forward (F) (dilution 1:10)
2.5 µlPrimer reverse (R) (dilution 1:10)

*green ones are the only specify.

IFN (1)IFN (2)AsLOVpep (1)AsLOVpep (2)
IFNIFNAsLOVpepAsLOVpep
Mys int FIFN domBB RAsLOVpep FAsLOVpep Fint
Mys int RIFN domBB FAsLOVpep RintAsLOVpep R
AsLOVpep (nested)PhiC31 (1)PhiC31 (2)PhiC31 (3)
AsLOVpepPhiC31PhiC31PhiC31
AsLOVpep nestedPhiC31 Fint 1PhiC31 Fint 2PhiC31 Fint 3
AsLOVpepPhiC31 Rint 2PhiC31 Rint 3PhiC31 R
PhiC31
PhiC31
PhiC31 F
PhiC31 Rint

Digestions of the minipreps:

PhiC31NotI2046, 1899
LacI:AsLOVpep+ePDZ+ren EcoRI6345, 8798
Gal4:AsLOVpep+ePDZ+ren EcoRI6345, 9569

Make the gel:

PhiC31 (C6)C7…C16LacI:AsLOVpep:ePDZ:ren (C1)C2C3…C4
nonooknook
Gal4:AsLOVpep+ePDZ+ren (C1)Gal4:AsLOVpep+ePDZ+ren (C2)
okok

Mesurement of the ODs:

Citoplasm0.217.35 ml
RepPhiC310.269.1ml
35S:LTB:T35S0.279.45ml
PhiC310.279.45ml
GFP0.2010ml
RepBxbI0.3311.55ml
PsinATG:RepBxbI:GFP0.3612.6ml
PhiC310.3411.9ml
DsRed0.269.1ml
P190.289.8ml

12 August 2015

New digestions to check if the negative control constructions are correct and we can transformed it in agro

OpLexA:luc:SF:renEcoRI6345, 7274
Op:UAS:luc:SF:renEcoRI6345, 7096
OpEtr8.luc:SF:renEcoRI6345, 7294

Gel:

Op:UAS:luc:SF:ren C1Op:UAS:luc:SF:ren C2Op:UAS:luc:SF:ren C3OpEtr8.luc:SF:ren
nononono
OpLexA:luc:SF:ren C1OpLexA:luc:SF:ren C2Partner dnaPartner dna
nono

Transform in Agro and make petri dishes cultures:

  • LexA:AsLOVpep:ePDZ
  • Gal4:AsLOVpep:ePDZ
  • LacI:AsLOVpep:ePDZ
  • LexA:PIF6:phyB:VP16
  • Gal4:PIF6:phyB:VP16
  • LacI:PIF6:phyB:VP16
  • All of them are in ?1.

Ligations:

LexA:AsLOVpep:ePDZ+ren; ?1Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1
1 µl LexA:AsLOVpep:ePDZ1 µl Gal4:AsLOVpep:ePDZ:ren1 µl LacI:AsLOVpep:ePDZ
1 µl renilla (159)1 µl OpUAS:luc1 µl OpLacI:luc
1 µl ?11 µl ?11 µl ?1
4.6 µl H2O4.6 µl H2O4.6 µl H2O